ABSTRACT
AIM:To clon human cytochrome P450 2A6 cDNA. METHODS:Using reverse transcription polymerase chain reaction(RT-PCR) and DNA recombinant technique, a full-length cDNA encoding cytochrome P450 2A6( CYP2A6 ) from human liver was cloned into pBluescript vector. The cDNA segment was identified by DNA sequencing.RESULTS: Comparing with the CYP2A6 sequence, the cloned CYP2A6 cDNA had two different bases, codon 8 CTG(Leu)→TTG(Leu), codon 479 GGC(Gly)→GTC(Val) and was quite different in their 3′end noncoding region. Comparing with CYP2A7 seqence reported by Fernandez-Salguero,the cloned CYP2A6 cDNA had some different in 5′ end coding sequence and several differencey in the 3′ end coding and noncoding sequence, but both codon 479 were GTC(Val).Comparing with the CYP2A7 seqence reported by Yamano,the cloned CYP2A6 cDNA had some difference in the coding sequence but the 3′ end no-coding area was the same. CONCLUSION: The cloned cDNA was a new cDNA of CYP2A6 which may be transcripted from a new allele of CYP2A6.
ABSTRACT
AIM:To demonstate the expression of human cytochrome P450 3A4 isozyme in the transgenic cell line CHL - 3A4 established in this laboratory. METHODS:Nifedipine (NIF) can induce the reversal of the brug resistance for adriamycin of multidrug resistant(MDR) cell K562r. The NIF is the specific substlate for CYP3A4. To determine the NIF oxidase activity of the transgenic CHL - 3A4 cells by comparing the biological effect of NIF, which preinculbated with or without CHL - 3A4 S9 mix. RESULTS: When the multidrug resistance cell K562r was cultured in medium with NIF (12. 5 ?g? L- 1 ), a calcium channel blocker and specific substrate for CYP3A, it's IC50 for adri- amycin declined from(6.47?0.60) mg ? L- 1 to (0. 89?0. 15) mg? L-1. When cells were cultured in NIF(exactly the same concentration ) pretreated with CHL - 3A4 S9 mix,no reversal of MDR was observed [IC50 for adriamycin is (6.10?0.50) mg? L-1 ] while if NIF was pretreated with CHL S9 or inactivated CHL - 3A4 S9 mix, its biological effect was not deteriorated [IC50 for adriamycin is (0. 32?0.90) and (0.32?0.04) mg?L-1, P